This amended proposal is a competitive renewal of a program that has as its goal the development of high throughput, high-resolution separation / mass spectrometry instrumentation for proteome analysis, using multiplex MALDI-MS and MS/MS. We call the approach - Capillary Array Deposition Mass Spectromtery - CADMS. While 2-D gel electrophoresis, followed by mass spectrometry, is well established for the analysis of the separated proteins, recent efforts in global proteomics have turned towards the use of shotgun techniques. In these shotgun techniques, large groups of proteins are isotopically labeled with either a light or heavy form, followed by pooling and digestion and then separation/analysis. Multidimensional separation is necessary because of the very large number of peptides that are formed. Analysis of these fractions can take days for completion at the present time. We will develop parallel processing high-resolution separations for MALDI-TOF. Multicolumn reversed phase LC separations of fractions from an initial cation exchange column will be deposited as narrow (250 micron wide) streaks on MALDI targets. These targets will then be analyzed on an MS instrument we propose to build, employing a 2 kHz repetition rate laser that will allow interrogation one or more orders of magnitude faster than presently possible. In addition, high mass resolution and accuracy will be characteristic of the instrument. Furthermore, new software will be developed to extract sample peaks from noise. Quantitative analysis wilt be achieved in the first round of MALDI-TOF MS. Subsequently, MS/MS sequence information will be obtained on those peaks, which are differentially expressed, using MALDI-TOF/TOF MS, again operating with the 2 kHz laser. New approaches to multiple precursor ion scanning will be implemented for even greater MS/MS candidate analysis. As one demonstration of our technology, we will examine the proteomic differences between normal and persistor E.coli cells, the latter being not eradicated when antibiotics are administered. As a second demonstration, we will conduct proteome analysis on a mammalian breast cancer cell line, SKBR-3. Both profiling and differential expression analysis will be undertaken. CADMS will significantly advance proteome characterization of human diseases.